Advancements in DNA Sequencing Techniques
Discover how linear PCR and current Sanger sequencing methods have revolutionized DNA amplification and sequencing processes, enabling efficient generation of terminated fragments with fluorescent dyes for accurate analysis and direct computer recording of DNA sequences in real-time.
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Presentation Transcript
1980s: Linear PCR is used to generate more products from less template Cycle sequencing is a simple method in which successive rounds of denaturation, annealing, and extension in a thermal cycler result in linear amplification of extension products. From applied biosystems.com
Current Sanger sequencing use dideoxy terminators that have different fluorescent dyes attached From Applied Biosystems Dyes are attached to the ddNTPs Only one reaction tube per sample, instead of four (isotope method) Fluorescent fragments are generated by incorporation of dye-labeled ddNTPs. Each different dideoxy nucleotide (ddATP, ddCTP, ddGTP, or ddTTP) carries a different colored dye. All terminated fragments (those ending with a ddNTP) contain a dye at their 3 end Only one lane/sample on gel, instead of four (isotope method)
Overview of procedure Tube contains all four fluorescently labelled dideoxynucleotides Linear PCR is used to generate multiple rounds of terminated fragments Sample is loaded in one capillary
The output of the detector can be sent directly to a computer and the DNA sequence recorded (in real time) as the gel is being run Example of output:
